Journal: International Journal of Biomaterials

Article Title: Enhancement of the Mechanical Properties of Hydroxyapatite/Sulphonated Poly Ether Ether Ketone Treated Layer for Orthopaedic and Dental Implant Application

doi: 10.1155/2018/9607195

Figure Lengend Snippet: SEM image of the surface of the treated layer (a) before and (b) after applying the compression load.

Article Snippet: Nanoscale morphology of the coated layer before and after compression and the morphology of the scratches were probed using a scanning electron microscope (SEM) (Hitachi Tabletop, TM-3000).

Techniques:

Journal: PLoS ONE

Article Title: 2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication

doi: 10.1371/journal.pone.0080769

Figure Lengend Snippet: (A) The expression of the CNP proteins was studied by immunofluorescence microscopy using the anti-CNP polyclonal rabbit antibody and Alexa Fluor 488-anti-rabbit IgG. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). (B) Huh7 and HepG2 cells were cultured without or with 300, 1000 IU/ml of IFNα-2b for 2 days. Cells were harvested and the cell lysates were determined by Western blot analysis. Actin served as a loading control (protein panel). (C) For CNP mRNA quantitative analysis, cDNA pools were synthesized from the 1 μg of total RNA of cells and diluted cDNA was amplified by quantitative real-time PCR. Actin was served as reverse transcription and PCR control. The mRNA% presents the ratio of values of total CNP (tCNP) to that of naive HepG2 cells (n=3).

Article Snippet: Membranes were blocked with 0.05% TBST (Tris-Buffered Saline Tween-20) containing 5% nonfat dry milk and probed with antibodies against CNP (Proteintech 1:3000), β-actin (Abcam, 1:5000), secondary antibodies conjugated to horseradish peroxidase were used and followed by ECL (Merck Millipore) detection.

Techniques: Expressing, Immunofluorescence, Microscopy, Cell Culture, Western Blot, Control, Synthesized, Amplification, Real-time Polymerase Chain Reaction, Reverse Transcription

Journal: PLoS ONE

Article Title: 2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication

doi: 10.1371/journal.pone.0080769

Figure Lengend Snippet: (A) Huh7 cells were transfected with pCDNA5-CNP1 or pCDNA5-CNP2 and harvested at day 2 post transfection. The expression of CNP and tubulin was revealed by immunofluorescence microscopy using the anti-CNP polyclonal rabbit antibody and anti-tublin monoclonal mice antibody, the secondary antibodies were Alexa Fluor 594-conjugated goat anti-rabbit IgG Alexa Fluor 488-conjugated goat anti-mouse or rabbit IgG. The nuclei were counterstained with DAPI. Mitochondria were stained using Mitocapture Tm kit and indicated by red color. White arrow indicates a CNP1-transfected Huh7 cell undergone a morphological change. (B) Huh7 ells in 12-well plate were transfected with 2 μg of plasmids pCDNA5-CNP1 or pCDNA5-CNP2. Cell lysates were determined by Western blot analysis. Actin served as a loading control.

Article Snippet: Membranes were blocked with 0.05% TBST (Tris-Buffered Saline Tween-20) containing 5% nonfat dry milk and probed with antibodies against CNP (Proteintech 1:3000), β-actin (Abcam, 1:5000), secondary antibodies conjugated to horseradish peroxidase were used and followed by ECL (Merck Millipore) detection.

Techniques: Transfection, Expressing, Immunofluorescence, Microscopy, Staining, Western Blot, Control

Journal: PLoS ONE

Article Title: 2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication

doi: 10.1371/journal.pone.0080769

Figure Lengend Snippet: Panels show micrographs of liver biopsy specimens from three HBV-infected patients. Specimens were costained with antibodies against HBV X protein (red) and CNP (green); nuclei were stained with DAPI (blue). These images were collected at 100 magnification and show the representative images. Control micrograph was collected from the non-infectious area. The dashed rectangle in P2 (Patient 2) indicated the HBx positive but CNP negative cells; the real line indicated the both positive area. The enlarge panel exhibits the subcellular distribution of HBx and CNP from the real line area of P3 panel.

Article Snippet: Membranes were blocked with 0.05% TBST (Tris-Buffered Saline Tween-20) containing 5% nonfat dry milk and probed with antibodies against CNP (Proteintech 1:3000), β-actin (Abcam, 1:5000), secondary antibodies conjugated to horseradish peroxidase were used and followed by ECL (Merck Millipore) detection.

Techniques: Infection, Staining, Control

Journal: bioRxiv

Article Title: Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

doi: 10.1101/773416

Figure Lengend Snippet: (A) PCR analyses of the Chlamydomonas fap76-1 , fap76-2 , fap81 , fap92 and fap216 mutants verified that the corresponding gene was disrupted by the insertion cassette. For each strain, the gene-specific primers could amplify the DNA fragments in wild type, but not in the mutant; in contrast, the cassette fragment could be amplified in the mutant, but not in the wild type. The primers used here are listed in Table S4. (B) RT-PCR analyses of the Chlamydomonas wild-type and fap92 mutant revealed that the mRNA of FAP92 was interrupted in the mutant. Red arrow indicates the right band of FAP92 cDNA in wild-type confirmed by sequencing and the white arrows indicate the non-specific bands that were not FAP92 cDNA fragment confirmed by sequencing. Beta-tubulin was used as the control gene. (C) Western blot analyses using anti-PF16 antibody and streptavidin-HRP show PF16 and BCCP-tagged PF16 in the axonemes of wild type and the rescued strains pf16;PF16::BCCP , respectively; the BCCP-tag adds 9 kDa to PF16. In contrast, PF16 was not detected in the pf16 mutant. The wild-type PF16 co-migrates with a large amount of tubulin and therefore the wild-type PF16 band typically appears of lower intensity than those of the larger PF16-BCCP in the rescued strains. Immuno-labeling of the radial spoke protein 1 (RSP1) was used as loading control. (D) Western blot analyses using anti-PF16 antibody showed that PF16 is assembled into the axonemes of the CLiP mutants fap76-1 , fap76-2 , fap81 , and fap216 and the rescued strain fap76-1;BCCP::FAP76 . Immuno-labeling of the radial spoke protein 1 (RSP1) was used as loading control.

Article Snippet: Cells were screened for rescued motility; incorporation of the construct was then confirmed by western blotting with a streptavidin-HRP probe (1:3000, Molecular Probes).

Techniques: Mutagenesis, Amplification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot, Immunolabeling

Journal: bioRxiv

Article Title: Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

doi: 10.1101/773416

Figure Lengend Snippet: (A-P) Tomographic slices (columns 1 and 2) and isosurface renderings (columns 3 and 4) of the averaged CA repeats of wild type (A-D), “9+2” pf16 (E-F), and of the tagged rescue pf16;PF16::BCCP without adding nanogold (I-L, control) and after treatment with streptavidin gold (M-P) in cross-sectional (columns 1 and 3) and longitudinal views (columns 2 and 4). The thin blue line in (A) indicates the location of the slices shown in column 2. The C1a, e and c projections (indicated by black brackets in A and C) were missing in the pf16 mutant (white bracket in E). When the C-terminus of PF16 was tagged with BCCP, the additional density of the BCCP-streptavidin-gold label was detected in the C1a projection (blue arrowheads in M-P); this label density was not observed in wild type (white arrowheads in A-D) or the control samples (white arrowheads in I-L). Scale bar in N, 20 nm (valid for all EM images).

Article Snippet: Cells were screened for rescued motility; incorporation of the construct was then confirmed by western blotting with a streptavidin-HRP probe (1:3000, Molecular Probes).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

doi: 10.1101/773416

Figure Lengend Snippet: (A) Immunofluorescence light microscopy images of axonemes isolated from fap76-1 (left) and fap76-1;BCCP::FAP76 rescue (right) probed by anti-acetylated-tubulin antibody (red) and fluorescently tagged streptavidin (green). The streptavidin signal indicates that the BCCP-tagged FAP76 was assembled into the axoneme of the rescued strain. (B) SDS-polyacrylamide gel stained by a silver enhancement kit (top) to show that a specific band of appropriate relative mobility could be detected in the fap76-1;BCCP::FAP76 axonemes treated with streptavidin-Au, but not in the control (-Au). Coomassie Brilliant Blue (CBB) staining (bottom) shows the tubulin bands as loading controls. (C) A classification analysis of the C1c peripheral subunit 1 (psu1/black arrows in E and F) in wild type and fap76-1 , and fap76-1;BCCP::FAP76 rescue axonemes (white arrows in H, I, K and L). The particle numbers (n) included in the averages for wild type, fap76-1 , fap76-1;BCCP::FAP76 (+Au) and fap76-1;BCCP::FAP76 (-Au) were 664, 927, 1089 and 935 (see Table S3). (D-L) Comparison of tomographic slices (D, E, G, H, J and K) and isosurface renderings (F, I and L) of the averaged CA repeats of wild type (D-F) vs. fap76-1;BCCP::FAP76 rescue strain either without (G-I) or treated with (J-L) streptavidin gold, viewed in cross-sectional (D, G and J) and longitudinal (E, F, H, I, K and L) orientations. The additional density of the streptavidin-gold label at the interface between the C1c and C1e projections in the gold-treated rescue strain (blue arrowheads in J-L) is not observed in wild-type or control CA (white arrowheads in D-I). Thin blue line in D indicates the location for the tomographic slices shown in E, H and K. Scale bar in A, 5 µm (valid for all fluorescence images); in K, 20 nm (valid for all EM images in D-L).

Article Snippet: Cells were screened for rescued motility; incorporation of the construct was then confirmed by western blotting with a streptavidin-HRP probe (1:3000, Molecular Probes).

Techniques: Immunofluorescence, Light Microscopy, Isolation, Staining, Fluorescence

Journal: Brain and Behavior

Article Title: In vivo overexpression of synaptogyrin‐3 promotes striatal synaptic dopamine uptake in LRRK2 R1441G mutant mouse model of Parkinson's disease

doi: 10.1002/brb3.2886

Figure Lengend Snippet: Adeno‐associated virus (AAV)‐mediated overexpression of SYNGR3 in striatum increased synaptosomal dopamine (DA) uptake. (a) Young 3‐month‐old wild‐type (WT) and LRRK2 mutant mice received intracranial injection of AAV7 (1 μl; injection rate: 0.1 μl/min) at one side of the brain hemisphere to induce SYNGR3 overexpression driven by Synapsin‐I promotor. WT and LRRK2 mutant mice were injected with AAV7‐SYNGR3 for 3 months before sacrifice. Diagram illustrates the design of the viral expression construct and the site of injection. (b) Overexpression of SYNGR3 on the injected side of the brain was confirmed by immunohistochemistry of SYNGR3 (red) compared to the opposite hemisphere of the same mouse brain. (c) Representative Western blots of two WT and two LRRK2 R1441G mutant mice showing overexpression of SYNGR3 in synaptosomal lysates extracted from injected mouse striatum 3 months after AAV injection. (d) Overexpression of SYNGR3 significantly increased striatal synaptosomal [3H]‐DA uptake, compared to the opposite non‐injected side of the striatum (paired t ‐test). There was no significant difference in synaptosomal DA uptake at non‐injected side of WT and LRRK2 mutant striatum. * p < .05 represents statistical significance between designated groups by paired t ‐test. Abbreviation: ns, not significant. R1441G, LRRK2 R1441G knockin mice

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane by electrophoresis in Tris‐Glycine transfer buffer (25 mM Tris, 190Mm glycine, and 15% methanol; pH 8.3) at 100 V for 2 h. The membrane was blocked with 5% non‐fat skimmed milk in TBS and probed with antibodies against SYNGR3 (1:3000, Santa‐Cruz Biotechnology; #sc‐271046, 26 kD), synaptophysin (SYP) (1:1000, Cell Signaling Technology #D35E4, 38 kD), DAT (1:1000, Santa‐Cruz Biotechnology #sc‐10042, 50 kD), and actin (1:3000, Santa‐Cruz Biotechnology #sc‐1615, 43 kD).

Techniques: Virus, Over Expression, Mutagenesis, Injection, Expressing, Construct, Immunohistochemistry, Western Blot, Knock-In

Journal: Brain and Behavior

Article Title: In vivo overexpression of synaptogyrin‐3 promotes striatal synaptic dopamine uptake in LRRK2 R1441G mutant mouse model of Parkinson's disease

doi: 10.1002/brb3.2886

Figure Lengend Snippet: Expression levels of SYNGR3 and synaptophysin (SYP) in striatum lysates from 3‐month‐old and 18‐month‐old wild‐type (WT) and LRRK2 R1441G mutant mice. (a) Quantification of SYNGR3 content in whole striatal lysates using a commercial ELISA based on a standard curve developed with a serial dilution of recombinant SYNGR3 protein standards supplied by the kit. (b) Illustrations of mouse brain dissection and isolation of striatum (STR) and cortex (CTX). (c) Representative Western blots of SYNGR3 and SYP in total striatal lysates. Densitometry analysis showed that SYNGR3 and SYP were significantly reduced in young LRRK2 mutant mice compared to WT control. Data are expressed as means ± SEM. (N = 11). * p < .05, ** p < .01, and *** p < .001 represent statistical significance between groups by Mann–Whitney (unpaired, nonparametric) test. Abbreviation: KI, knockin

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane by electrophoresis in Tris‐Glycine transfer buffer (25 mM Tris, 190Mm glycine, and 15% methanol; pH 8.3) at 100 V for 2 h. The membrane was blocked with 5% non‐fat skimmed milk in TBS and probed with antibodies against SYNGR3 (1:3000, Santa‐Cruz Biotechnology; #sc‐271046, 26 kD), synaptophysin (SYP) (1:1000, Cell Signaling Technology #D35E4, 38 kD), DAT (1:1000, Santa‐Cruz Biotechnology #sc‐10042, 50 kD), and actin (1:3000, Santa‐Cruz Biotechnology #sc‐1615, 43 kD).

Techniques: Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Serial Dilution, Recombinant, Dissection, Isolation, Western Blot, Control, MANN-WHITNEY, Knock-In

Journal: Brain and Behavior

Article Title: In vivo overexpression of synaptogyrin‐3 promotes striatal synaptic dopamine uptake in LRRK2 R1441G mutant mouse model of Parkinson's disease

doi: 10.1002/brb3.2886

Figure Lengend Snippet: SYNGR3 co‐localizes and interacts with dopamine uptake transporter (DAT) in striatum. (a) Immunohistochemistry showed co‐localization of SYNGR3 (red) and DAT (green) in dorsal striatal region of mouse brain. Area of SYNGR3‐DAT colocalization was identified using Adobe Photoshop™, which were shown as white puncta in the right panel. (b and c) Immunogold staining under transmission electron microscopy (TEM) demonstrated spatial proximity between SYNGR3 and DAT in striatum. Multiple snapshots show SYNGR3‐DAT co‐localization in striatal pre‐synaptic termini; magnification: 1:245,000. (d) Immunoprecipitation of DAT using anti‐DAT antibody resulted in co‐precipitation of SYNGR3 from wild‐type (WT) (i) and LRRK2 R1441G mutant (ii) mouse brain striatal lysates, and (ii, iv) vice versa. Target bands are highlighted by the rectangular boxes.

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane by electrophoresis in Tris‐Glycine transfer buffer (25 mM Tris, 190Mm glycine, and 15% methanol; pH 8.3) at 100 V for 2 h. The membrane was blocked with 5% non‐fat skimmed milk in TBS and probed with antibodies against SYNGR3 (1:3000, Santa‐Cruz Biotechnology; #sc‐271046, 26 kD), synaptophysin (SYP) (1:1000, Cell Signaling Technology #D35E4, 38 kD), DAT (1:1000, Santa‐Cruz Biotechnology #sc‐10042, 50 kD), and actin (1:3000, Santa‐Cruz Biotechnology #sc‐1615, 43 kD).

Techniques: Immunohistochemistry, Staining, Transmission Assay, Electron Microscopy, Immunoprecipitation, Mutagenesis

Journal: Brain and Behavior

Article Title: In vivo overexpression of synaptogyrin‐3 promotes striatal synaptic dopamine uptake in LRRK2 R1441G mutant mouse model of Parkinson's disease

doi: 10.1002/brb3.2886

Figure Lengend Snippet: Overexpression of SYNGR3 increased cellular dopamine (DA) uptake in human SH‐SY5Y neuroblastoma cells. (a) Immunocytochemistry showing that SYNGR3 tagged with green fluorescent protein (GFP) (green) was overexpressed and co‐localized with synaptophysin (SYP; synaptic vesicle marker protein; red) in human SH‐SY5Y cells; magnification: 1:630. The lower panels showed magnified views of transfected cells in the dashed box. (b) Western blot analysis of total cell lysates after transient overexpression of SYNGR3 compared to empty‐vector controls. (c) Overexpression of SYNGR3 in SH‐SY5Y cells caused significant increase in total cellular [3H]‐DA uptake activity compared with empty‐vector controls. (d) Flow cytometry assay after whole cell immunostaining of cell surface DAT. Mean cellular fluorescent intensity was calculated based on measurements of 20,000 cells in each group. SYNGR3 overexpression did not affect levels of cell surface DAT. * p < .05, and *** p < .001 represent statistical significance between groups by unpaired Student's t ‐test.

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane by electrophoresis in Tris‐Glycine transfer buffer (25 mM Tris, 190Mm glycine, and 15% methanol; pH 8.3) at 100 V for 2 h. The membrane was blocked with 5% non‐fat skimmed milk in TBS and probed with antibodies against SYNGR3 (1:3000, Santa‐Cruz Biotechnology; #sc‐271046, 26 kD), synaptophysin (SYP) (1:1000, Cell Signaling Technology #D35E4, 38 kD), DAT (1:1000, Santa‐Cruz Biotechnology #sc‐10042, 50 kD), and actin (1:3000, Santa‐Cruz Biotechnology #sc‐1615, 43 kD).

Techniques: Over Expression, Immunocytochemistry, Marker, Transfection, Western Blot, Plasmid Preparation, Activity Assay, Flow Cytometry, Immunostaining

Journal: Brain and Behavior

Article Title: In vivo overexpression of synaptogyrin‐3 promotes striatal synaptic dopamine uptake in LRRK2 R1441G mutant mouse model of Parkinson's disease

doi: 10.1002/brb3.2886

Figure Lengend Snippet: Effects of SYNGR3 expression on innate marble burying behavior. (a) Young (3 months old) wild‐type (WT) and LRRK2 R1441G mutant receiving stereotaxic injection of either adeno‐associated virus‐7–green fluorescent protein (AAV7–GFP) or AAV7‐SYNGR3 were subjected to marble burying test at 3 months after AAV injection. A total of 15 marbles were evenly distributed on the bedding inside the cage in form of a 5 × 3 matrix. (b) The number of marbles which have been covered two‐thirds by bedding were counted at 15 and 30 min. Total number of marbles buried (at 30 min) by LRRK2 mutant mice were significantly increased after overexpressing SYNGR3 in the striatum. Data are expressed as means ± SEM. (N = 5). * p < .05 and ** p < .01 represent statistical significance between the two designated groups by unpaired, Student's t ‐test.

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane by electrophoresis in Tris‐Glycine transfer buffer (25 mM Tris, 190Mm glycine, and 15% methanol; pH 8.3) at 100 V for 2 h. The membrane was blocked with 5% non‐fat skimmed milk in TBS and probed with antibodies against SYNGR3 (1:3000, Santa‐Cruz Biotechnology; #sc‐271046, 26 kD), synaptophysin (SYP) (1:1000, Cell Signaling Technology #D35E4, 38 kD), DAT (1:1000, Santa‐Cruz Biotechnology #sc‐10042, 50 kD), and actin (1:3000, Santa‐Cruz Biotechnology #sc‐1615, 43 kD).

Techniques: Expressing, Mutagenesis, Injection, Virus

Journal: Brain and Behavior

Article Title: In vivo overexpression of synaptogyrin‐3 promotes striatal synaptic dopamine uptake in LRRK2 R1441G mutant mouse model of Parkinson's disease

doi: 10.1002/brb3.2886

Figure Lengend Snippet: Schematic diagram illustrating how SYNGR3 expression level can affect dopamine (DA) uptake and sequestration into synaptic vesicles via interaction between SYNGR3 and DAT. Egana et al. first reported interaction between SYNGR3 and DAT in the vesicular DA storage system. Induced expression of SYNGR3 on synaptic vesicle surface can recruit vesicles into close proximity to dopamine uptake transporter (DAT) near the plasma membrane for more efficient and rapid sequestration of cytosolic free DA into synaptic vesicles via VMAT‐2.

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane by electrophoresis in Tris‐Glycine transfer buffer (25 mM Tris, 190Mm glycine, and 15% methanol; pH 8.3) at 100 V for 2 h. The membrane was blocked with 5% non‐fat skimmed milk in TBS and probed with antibodies against SYNGR3 (1:3000, Santa‐Cruz Biotechnology; #sc‐271046, 26 kD), synaptophysin (SYP) (1:1000, Cell Signaling Technology #D35E4, 38 kD), DAT (1:1000, Santa‐Cruz Biotechnology #sc‐10042, 50 kD), and actin (1:3000, Santa‐Cruz Biotechnology #sc‐1615, 43 kD).

Techniques: Expressing, Clinical Proteomics, Membrane

Journal: The Journal of Neuroscience

Article Title: Dendritic Ca 2+ -Activated K + Conductances Regulate Electrical Signal Propagation in an Invertebrate Neuron

doi: 10.1523/JNEUROSCI.19-19-08319.1999

Figure Lengend Snippet: Voltage-gated Ca2+ channels are present in the AP cell neurites. A, Morphology of the AP cell. Light-microscopic image of the AP cell filled with fluorescein after brightness and contrast adjustment and thresholding. The outline of the ganglion is indicated by the line drawing. Anterior is up. Scale bar, 100 μm. B, Increases in the intracellular [Ca2+] in the soma evoked by depolarization to −25 mV via somatic current injection (+2 nA) from a holding potential of −45 mV in normal leech saline and in 0 Ca2+, 1.8 mm Co2+saline. The ganglion was cut at the midline to avoid the occurrence of Na+ spikes; average of two trials. C,Increases in the intracellular [Ca2+] at various neurite locations and in the soma evoked by a depolarization (+3 nA) from a holding potential of −60 mV in 10 mm TEA saline in the intact ganglion. All traces are normalized to their maximum value to facilitate the comparison of the time course. Inset,Fluorescent image of the AP cell filled with Calcium Green 1. Theopen boxes indicate the positions for the traces in the main figure. Scale bar, 100 μm. D, Repetitive Ca2+ spikes with Na+ spikes riding on the plateaus evoked by a depolarization via current injection (+3 nA) from a holding potential of −60 mV in the intact ganglion in saline containing 10 mm TEA. The fluorescent signal was monitored from the major neurite close to the midline.Inset, Schematic of the AP cell and outline of the ganglion. The location of the optical recording site is indicated by the gray spot near the midline. E, Repetitive Ca2+ spikes evoked by a depolarization via current injection (+2 nA) from a holding potential of −60 mV in the truncated ganglion in saline containing 10 mm TEA. The fluorescent signal was monitored from the major neurite close to the midline. Inset, Schematic of the AP cell and outline of the ganglion. The location of the optical recording site is indicated by the gray spot near the midline, and the location of the cut is indicated by the white bar near the midline.F, Voltage dependence of increases in intracellular [Ca2+]. Average peak change in fluorescence in the soma in response to a depolarization from a holding potential of −60 mV to various test membrane potentials via 500-msec-long somatic current injections in normal saline (mean ± SEM;n = 5 cells). The ganglion was cut at the midline to avoid Na+ spikes. Optical data from each experiment were normalized to their value at −30 mV, and test membrane potentials caused by current injection were binned (5 mV bin width).Inset, Increases in intracellular [Ca2+] in the soma evoked by a depolarization from a holding potential of −60 mV to various test potentials via somatic current injection in normal leech saline for one representative cell. The bottom trace indicates the timing of the current injection.

Article Snippet: To observe AP cells in light microscopy, an AP cell was iontophoretically injected with fluorescein dextran (5% w/v in H 2 O; 3000 molecular weight; Molecular Probes, Eugene, OR) using sharp electrodes of 10–30 MΩ resistance and pulsed current (−7 to −3 nA; 10 Hz; 30 min).

Techniques: Injection, Saline, Comparison, Fluorescence, Membrane

Journal: Cells

Article Title: Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset

doi: 10.3390/cells10112939

Figure Lengend Snippet: Cav-1 levels increase as cells reach senescence. ( A ) Characteristic Immunoblot showing caveolin-1 (Cav-1) levels increasing significantly as WJ-MSCs reach senescence. β-Actin was used as control for equal protein loading. ( B ) Representative photos of early-, middle- and late-passage cells stained with SA-β-gal staining solution. ( C ) Graph showing the percentage of SA-β-gal positive cells in early-, middle- and late-passage cells as mean from three donors. * p < 0.05 vs. NT or otherwise indicated. p values calculated using the Student t -test. ( D ) Representative images of early- and late-passage WJ-MSCs where total ROS and superoxide levels were analyzed under normal conditions (early–late). For a positive control pyocyanine was added to the early passage cells (early + PYO). Images were captured with the 40X objective lens of the fluorescent microscope used.

Article Snippet: Membranes were further probed with an antibody against β-actin (1:3000 dilution, Mouse, Santa Cruz Biotechnology Inc.), which served as a loading control.

Techniques: Western Blot, Control, Staining, Positive Control, Microscopy

Journal: Cells

Article Title: Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset

doi: 10.3390/cells10112939

Figure Lengend Snippet: No alterations in Cav-1/pCav-1 levels in late-passage cells after exogenous oxidative insult. ( A ) Representative immunoblot of cell lysates of early- and late-passage WJ-MSCS (from three donors) that have been exposed to H 2 O 2 (300 μΜ, 30 min) and left to recover for 24 h, analyzed at four different time points (NT(=No Treatment), 1 h, 3 h, and 24 h of recovery time post-treatment) by western blotting using the anti-caveolin-1 (Cav-1) antibody. ( B ) Quantification of relative protein expression levels of Cav-1 in cell lysates from early and late WJ-MSCs (from 3 donors) after being exposed to H 2 O 2 was performed based on band density using ImageJ. NT values from both early- and late-passage cells were arbitrarily set to 1. Values shown are the means ± S.E. * p < 0.05 vs. the NT condition. ( C ) Characteristic images of early- and late-passage cells where Cav-1 localization was evaluated by staining with anti-cav-1 antibody and the appropriate secondary antibody (red) under normal conditions (No Treatment—NT) and 24 h after being treated with Doxorubicin. DNA damage was also assessed by staining with the 53BP1 antibody and the appropriate secondary antibody (green). Nuclei were stained with DAPI (blue). Images were captured with the 40× objective lens of the fluorescent microscope used. ( D ) Percentages of WJ-MSCs with 53BP1 foci from early- and late-passage cells from three donors evaluated by staining with the 53BP1 antibody under normal conditions (No Treatment—NT) and 24 h after Doxorubicin treatment. Values shown are the means ± S.E. # p < 0.05 vs. early passage cells at NT and * p < 0.05 vs. NT or otherwise indicated. p values calculated using the Student t -test. ( E ) Representative immunoblot of cell lysates of early- and late-passage WJ-MSCS (from 3 donors) that have been exposed for 24 h to Doxorubicin using anti-caveolin-1 (Cav-1) antibody. ( F ) Quantification of relative protein expression levels of Cav-1 in cell lysates from early and late WJ-MSCs (from 3 donors) after being exposed to 24 h Doxorubicin was performed based on band density using ImageJ. NT values from both early- and late-passage cells were arbitrarily set to 1. Values shown are the means ± S.E. * p < 0.05 vs. the NT condition. ( G ) Representative immunoblot showing p-Cav-1 levels from cell lysates of early- and late-passage WJ-MSCS (from three donors) that have been exposed to H 2 O 2 (300 μΜ, 30 min) and left to recover for 24 h, analyzed at three different time points ((NT(=No Treatment), 1 h and 24 h of recovery time post-treatment). Antibody against β-Actin was used as loading control for all western blots (A, B, D). ( H ) Quantification of relative protein expression levels of p-Cav-1 in cell lysates from early and late WJ-MSCs (from three donors) after being exposed to H 2 O 2 was performed based on band density using ImageJ. Values shown are the means ± S.E. * p < 0.05 vs. the NT condition and # p < 0.05 vs. early passage cells. p values were calculated using Student t -test.

Article Snippet: Membranes were further probed with an antibody against β-actin (1:3000 dilution, Mouse, Santa Cruz Biotechnology Inc.), which served as a loading control.

Techniques: Western Blot, Expressing, Staining, Microscopy, Control

Journal: Cells

Article Title: Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset

doi: 10.3390/cells10112939

Figure Lengend Snippet: Cav-1 levels increase in SIPS. ( A ) Representative photos of early passage (early) cells untreated (early) and after being subjected to stress induced premature senescence (SIPS) by treatment with 400 μΜ of H 2 O 2 , 2 h at passage 2 (SIPS p2) stained with SA-β-gal staining solution. ( B ) Graph showing the percentage of SA-β-gal positive cells in early and SIPS p2 cells. Values shown are the means ± S.E. * p < 0.05 vs. early passage WJ-MSCs. ( C ) Characteristic immunoblot showing caveolin-1 (Cav-1) levels increasing significantly in SIPS p2 cells. Antibody against β-Actin was used as loading control. ( D ) Graph demonstrating Cav-1 levels in early and SIPS p2 cells, based on band density calculated using Image J. Values shown are the means ± S.E. * p < 0.05 vs. the early passage WJ-MSCs.

Article Snippet: Membranes were further probed with an antibody against β-actin (1:3000 dilution, Mouse, Santa Cruz Biotechnology Inc.), which served as a loading control.

Techniques: Staining, Western Blot, Control

Journal: Cells

Article Title: Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset

doi: 10.3390/cells10112939

Figure Lengend Snippet: Cav-1 downregulation results in failure of oxidative DNA damage repair. ( A ) Characteristic immunoblot showing caveolin-1 (Cav-1) levels decreasing significantly after siRNA against Cav-1 treatment in early passage cells. Antibody against β-Actin was used as loading control. ( B ) Graph demonstrating Cav-1 levels based on band density calculated using Image J. Values shown are the means ± S.E. * p < 0.05 vs. the early passage WJ-MSCs treated with control siRNA (siCTRL). ( C ) Characteristic images of early passage cells treated with control siRNA and siRNA against Cav-1, under normal conditions (No Treatment—NT) and at 1 and 24 h after being treated with H 2 O 2 (300 μΜ, 30 min) stained with anti-cav-1 antibody and the appropriate secondary antibody (red). DNA damage was also assessed by staining with the 53BP1 antibody and the appropriate secondary antibody (green). Nuclei were stained with DAPI (blue). Images were captured with the 40× objective lens of the fluorescent microscope used. ( D ) Percentages of cells with 53BP1 foci from early passage WJ-MSCs treated with siCTRL and siCav-1 under normal conditions (No Treatment—NT) and at 1 h and 24 h after being treated with H 2 O 2 (300 μΜ, 30 min). Values shown are the means ± S.E. # p < 0.05 vs. siCTRL treated early passage cells and * p < 0.05 vs. NT or otherwise indicated. p values calculated using the Student t -test.

Article Snippet: Membranes were further probed with an antibody against β-actin (1:3000 dilution, Mouse, Santa Cruz Biotechnology Inc.), which served as a loading control.

Techniques: Western Blot, Control, Staining, Microscopy

Journal: Cells

Article Title: Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset

doi: 10.3390/cells10112939

Figure Lengend Snippet: Cav-1 function in senescence is mediated by autophagy. ( A ) Characteristic immunoblot showing Atg5 levels decreasing significantly after siRNA against Atg5 treatment in early passage cells. Antibody against β-Actin was used as loading control. ( B ) Graph demonstrating Atg5 levels based on band density calculated using Image J. Values shown are the means ± S.E. * p < 0.05 vs. the early passage WJ-MSCs treated with control siRNA (siCTRL). ( C ) Representative photos of early passage WJ-MSCs treated with siRNA control (siCTRL), siRNA against Atg5 (siAtg5), siRNA against caveolin-1 (siCav-1), and early passage WJ-MSCs treated with double siRNA against caveolin-1 (siCav-1) and against siAtg5 (siCav-1+siAtg5) stained with SA-β-gal staining solution. ( D ) Graph showing the percentage of SA-β-gal positive cells in early passage WJ-MSCs treated with siRNA control (siCTRL), siRNA against Atg5 (siAtg5), siRNA against caveolin-1 (siCav-1), and early passage WJ-MSCs treated with double siRNA against caveolin-1 (siCav-1) and against siAtg5 (siCav-1+siAtg5). p values shown are the means ± S.E. * p < 0.05 vs. siCav-1. ( E ) Characteristic immunoblot showing protein levels of key molecules of autophagy (ULK1, Atg13, PI3K-III, Beclin-1, Atg5, p62, LC3-I/II) in early passage cells treated with control siRNA (siCTRL) or siRNA against Cav-1 (siCav-1). Antibody against β-Actin was used as loading control.

Article Snippet: Membranes were further probed with an antibody against β-actin (1:3000 dilution, Mouse, Santa Cruz Biotechnology Inc.), which served as a loading control.

Techniques: Western Blot, Control, Staining